Effects of supplementation of pomegranate processing by-products and waste cooking oils as alternative feed resources in broiler nutrition

Agricultural residues can be used as alternative feed sources in industrial chicken production. The impacts of different levels of pomegranate peel and waste cooking oil as an agricultural residue on broilers' nutrition were investigated. Results showed that the replacement of 8% pomegranate peel in diets decreased the growth performance of broilers. Supplementing 8% pomegranate peel in diets reduced apparent nutrient digestibility. The highest level of waste oil inclusion in broiler diets indicated negative impacts on apparent zmetabolizable energy and crude fat apparent nutrients digestibility. Broilers fed the diet containing 4% pomegranate peel had a higher Lactobacillus population. The results showed that the Lactobacillus population was lower in broilers fed 8% pomegranate peel powder and 4% waste oil in diets. The inclusion of 8% pomegranate peel powder in diets showed lower villus height and crypt depth in the duodenum, jejunum, and ileum. The inclusion of 4% pomegranate peel decreased the peroxide value (PV) of meat. Dietary inclusion of 4% waste oil raised the PV of meat. Alpha-tocopherol supplementation decreased the PV of meat. Finally, the results provide information that 4% of pomegranate peel and 4% waste oil could be used as an alternative feed ingredient in broiler diets without adverse effects.

www.nature.com/scientificreports/ had an antiviral potential. In this regard, the researchers demonstrated that pomegranate peel could prevent SARS-CoV-2 entry into host cells. Also, the researchers showed that pomegranate peel polyphenols had antiviral impacts against COVID-19 8 . The effectiveness of these compounds and especially polyphenols (tannins) and saponins were investigated for their antimicrobial activity and their effectiveness in gastrointestinal tract improvement 9 . Waste vegetable oil (oxidized oil), obtained from food processing which is mostly produced due to lifestyle changes and increased consumption of fast food by human beings, may be used as a high-energy, and low-cost feed resource in broiler diets 2 . Some previous studies demonstrated that the waste oil could be supplemented in diets without any adverse effects for broilers. In contrast, some others observed that utilization of high peroxide value oil (oxidized oil) in diets has disadvantageous impacts on the health and performance of birds 10 . It seems that the supplementation of waste oil resulted in increased free radicals and oxidative stress in birds. It should be noted that malondialdehyde (MDA) is the second product of peroxidation that has a harmful influence on broiler health 11 . In general, waste oil generates free radicals which could have harmful consequences on the intestinal performance and health of broilers 12 . Reactive oxygen species (ROS) resulted in oxidative stress in the body, which harms broiler health and digestibility 11 . Pomegranate peel contains phenolic compounds such as tannins which can have antioxidant activity in broilers 13 . Also, it seems that the antioxidant characteristics of pomegranate peel reduced the adverse impacts of oxidative stress caused by oxidized oil 4 . The improvement in the global populations has caused a global revolution in food consumption models and enhanced requests for livestock products such as broiler chickens 14 . The higher demands in human life have led to a direct effect on increasing demand and production of livestock feedstuff which has inevitably placed increased pressure on limited resources 14 . Scientists indicated that pomegranate peel and waste vegetable oil as a by-product can replace in broiler diets. It seems that consuming this waste in poultry nutrition can diminish the disposal of this waste in the environment thus preventing environmental pollution. Pomegranate peel can also be a potential source of raw material for the animal feed industry. Generally, it has been hypothesized that utilizing these products can result in decreasing waste production and broiler industry diet cost. Also, it seems that pomegranate peel is one of the medicinal plants that had a strong antioxidant activity for poultry 2 . Hence, pomegranate peel and oxidized oil may play an important function as a new resource for broiler diets 15 . Nevertheless, a general investigation of the effects of these products on intestinal function and digestibility seems important. Therefore, the present study aimed to investigate the effects of using waste oils and pomegranate peel in diets on growth performance, intestinal function, apparent nutrient digestibility, and meat quality in broiler chickens.

Materials and methods
Animals, breeding, and nutrition. The research protocol has been approved by the Animal Care and Use Committee of the Islamic Azad University (93/987-2014) 16 , The research protocol advises animal rights and welfare by ensuring minimal stress to animals. All procedures were carried out by the relevant research protocol. The experiment was carried out in keeping with the ARRIVE guidelines. The experiment was designed according to 3 × 3 × 2 factorial arrangement, with factors (i) the pomegranate peel powder (0, 4, and 8% in diets), (ii) the oxidized oil (0, 2, and 4% in diets) and (iii) the α-tocopherol (zero and 200 mg/kg). Hence, a total of 1080-day-old, male Ross 308 strain broiler chickens, were randomly allocated to 90-floor pens in a completely randomized design with 18 treatments and five replicates with twelve chicks in each replicate. The chicks were fed commercial diets during the first 10 days of the experiment. At the end of 10 days, the chicks were weighed and housed in 1.5 × 1.5 m floor pens each equipped with a pan feeder and manual drinker. Stocking density is based on the EU broiler welfare Directive (2007), 33 kg/m 2 . Birds received 22L:2D throughout the experiment period. Feed and water were available ad libitum. In this study, chicks received mash diets. All chicks were kept under management conditions according to the Ross 308 strain catalog. All chicks in the treatment groups were fed a grower (11-24 days) and finisher diet (25-49 days). All diets were formulated based on nutritional requirements proposed by the National Research Council 17 (Tables 1 and 2). The composition (CP EE and CF) of all diets was measured by the AOAC method 18 (Table 3). The basal diets in this experiment were corn and soybean meal and according to the treatments, different percentages of pomegranate peel powder, oxidized soybean oil, and α-tocopherol were added to the basal diets. The commercial α-tocopherol in this experiment was provided by Science Laboratories ® (Info@Sclabs.ir), Iran, and is a powder α-tocopherol supplement that consists of 5500 IU α-tocopherol.
Pomegranate peel composition. Pomegranate peel was obtained from a pomegranate processes factory.
Peel was dried under ambient conditions and then milled and prepared for the experiment. In addition, the proximate chemical composition of pomegranate peel (i.e. dry matter (DM), crude protein (CP), Ether extract (EE), crude fiber (CF), nitrogen-free extract (NFE), and ash) was measured according to AOAC 18 methods. Furthermore, hydrolysable tannins and condensed tannins in pomegranate peel were obtained 15 . The essential amino acids in pomegranate peel such as methionine, lysine, and threonine were analyzed according to the method defined by the AOAC 18 (2005) and Cooperative 19 using HPLC and the modification of the PICO-TAG process. The chemical composition of pomegranate peel is shown in Table 2. The gross energy of the pomegranate peel was measured by an adiabatic bomb and is shown in Table 1 20 . The Sib bald method was used to determine the metabolizable energy of pomegranate peel 21 (Table 4).

Analyzed composition of diets
Gut morphology. On the last day of the experiment, and before slaughtering the birds, all of them starved for five hours and the feeding troughs were removed from the pens. Two broilers from each pen was slaughtered and subsequently, 5 cm length of intestine was sampled from the duodenum, jejunum, and ileum of it. The intestine samples were washed with sodium phosphate buffer and fixed by Clark stabilizer solution. All of the intestine samples were prepared for microscopic investigation later staining with Periodic Acid Schiff Solution  www.nature.com/scientificreports/ by separating the muscular layer and making the lamella. Ultimately, the villi height and the crypt depth were estimated according to the process of Ghasemi-Sadabadi et al. 6 .
Meat quality. At the end of the experiment, two broilers from each pen were slaughtered and the meat was trimmed and prepared for fatty acid profile and peroxide value measurement. The breast muscle was trimmed and stored at − 20 °C. Next, the meat sample (50 g) was mixed in a blender with 150 ml of Chloroform-methanol (Merck) in a 1:2 ratio for a minute. Then, 50 mL of sodium chloride (0.88%) was added to the mixture. Potassium chloride was used for dewatering, the aqueous phase was collected and methanol-potassium chloride 0.88% (v/v: 1/1) was mixed with the aqueous phase. The final solution was dried at 35 °C, after that, the residual solvent was evaporated under nitrogen gas pressure to obtain pure oil. Eventually, the fat was obtained for the measurement of peroxidation. To measure meat peroxidation, chloroform-acetic acid (25 ml) was added to one g of extracted fat in a ratio of 2:3. Then, one mL of potassium iodide was added to the mixture, which was stored for five min in the dark. The mixture was tittered with normal 1% potassium thiosulfate 6 . Peroxidation value was calculated based on the amount of peroxide (meq/kg extracted fat), according to the American Oil Chemists Society 22 . The fatty acid profile of the meat was analyzed by gas chromatography of the method as described by Ghasemi-Sadabadi 6,27 .
Statistical analysis. Statistical analyses were performed using SAS (2003) software. The data were analyzed using a 3 × 3 × 2 factorial arrangement design. Significant differences between treatments were separated using the Tukey range test at (p < 0.05). Data following model: Y ijk is the dependent variable; μ is the overall mean; P i is the effect of different pomegranate peel; O j is the effect of waste oil; V k of is the effect of α-tocopherol; P i O j is the interaction of pomegranate peel and waste oil; P i V k is the interaction of pomegranate peel and α-tocopherol; O j V k is the interaction of waste oil and α-tocopherol; P i O j V k is the interaction of pomegranate peel, waste oil, and α-tocopherol; E ijk is the random error.

Results and discussions
Performance. The effects of using different levels of pomegranate peel, waste soybean cooking oil, and α-tocopherol in the diet on BWG, FI, and FCR in broiler chickens are shown in Table 6. The results of this research revealed that the use of pomegranate peel in diets had significant influences on BWG, FI, and FCR (p < 0.05). The data showed that the dietary addition of 8% pomegranate peel in broiler diets decreased BWG and FI compared to other groups (p < 0.05). No significant differences were observed in growth performance between the 4% pomegranate peel and the control group. In this experiment, growth performance during 11-49 days was not significantly affected by waste oil and α-tocopherol (p < 0.05). No significant interaction between pomegranate peel, waste oil, and α-tocopherol on growth performance results has been observed in this experiment. By examining the results obtained, it can be concluded that when incorporating 8% pomegranate peel into diets, the growth performance of broilers decreased in the experimental period. In similar experiments, researchers confirmed that the inclusion of pomegranate peel in diets diminished the performance of birds 4,15 .
Similarly, the researchers stated that the addition of three g/kg pomegranate peel in broilers' diets decreased the growth performance 4,15 . One of the results of our research was that the inclusion of 4% pomegranate peel in diets had not significantly affected the growth performance of broilers. Also, Abbas et al. 5 indicated that the addition of 4.5 and 7.5% pomegranate peel inclusion in diets did not affect the growth performance of Japanese quail. Generally, it seems that high amounts of tannins and CF of pomegranate peel impaired the growth performance of the birds 4 . Furthermore, Ghasemi-Sadabadi et al. 2 stated that the pomegranate peel compounds such as CF and tannins may reduce gastrointestinal motility and consequently decrease the growth performance of birds. It has already been determined that the high amount of tannic acid reduces BWG and FI in poultry 28 . Past studies have shown that tannins can affect the physiological and biochemical systems of the body. In addition, tannins in the body lead to adverse nitrogen balances, growth inhibition, diminished intestinal absorption of sugars and amino acids, reduced immune response, and improved protein catabolism 29 . In this study, α-tocopherol did not affect growth performance. Contrary to this result, researchers indicated that vitamin E may be used in diets containing up to 6% of Distilled fatty acids for male quails because of the resulting betterment in nutrient utilization, growth performance, and meat quality 30 .
Apparent nutrient digestibility. The results (Table 7) indicated that dietary inclusion of 8% pomegranate peel in broiler diets decreased the digestibility of DM, CP, CF, and apparent metabolizable energy (AME) compared to other groups (p < 0.05). In this study, the results demonstrated that broilers fed diets with 4% waste oil had significantly lower digestibility of CF and AME when compared to the control group (p < 0.05). There was no significant difference in nutrient digestibility between the control group and 2% waste oil treatments. According to the results, no significant interaction between treatments on results was observed in this experiment. In this study, DM, CP, CF, and AME digestibility of broilers decreased when incorporating 8% pomegranate peel into diets. The results were quite similar to the results of Saleh et al. 15 ; Ghasem-Sadabadi et al. 2 . Also, our results agree with Akuru et al. 31 , who stated that the addition of pomegranate peel in diets reduced DM, CP, and CF digestibility in broilers. Similar effects were discussed by Saleh et al. 32 for the nutrient digestibility of broilers fed high dietary levels of pomegranate peel. Consequently, it appears that the lower digestibility in the 8% pomegranate peel group in this study could be related to higher tannins and fiber contents 32 . Furthermore, previous examina- www.nature.com/scientificreports/ tions confirmed that the pomegranate peel contains a high amount of CF and tannin, which adversely affected absorption and digestion 33 . The high amount of tannins had destructive influences on the physiological and biochemical systems, which resulted in adverse nitrogen balances, lower intestinal absorption of sugars and amino acids, diminished immune response, and increased protein catabolism 29 . It has been mentioned that phenolic compounds such as tannins could be bonded to dietary and endogenous proteins such as digestive enzymes and proteins located in the gastrointestinal tract that reduced the apparent nutrient digestibility (Brenes et al. 34 ). The reaction between the hydroxyl groups of polyphenol and the carbonyl groups of protein can explain the lower Table 6. The effects of using pomegranate peel, waste oil and α-tocopherol in diets on growth performance in broiler chickens (11-49 days). SEM standard error of mean, P-value probability values. a :b Means in columns with the same superscript do not differ significantly P < 0.05.

Main effect
Pomegranate peel (%) www.nature.com/scientificreports/ digestibility of protein in this study 35 . Similarly, researchers concluded that the use of high-tannin sorghum grain in broiler diets reduced protein digestibility 34 . The earlier reports had shown that the use of high polyphenol compounds in diets decreased fat absorption in rats and birds. Generally, tannins could bind to bile salts and cholesterol and reduce the digestibility of fat. Furthermore, trypsin, amylase, and lipase are the main digestive enzymes in birds that can be bonded by tannins and reduced digestibility of nutrients 32 . The results reveal a clear significant correlation between the waste oil and digestibility. According to the results, the supplementation of 4% waste oil decreased fat and AME digestibility. Our findings are in agreement with those of Kamran et al. 36 , Table 7. The effects of using pomegranate peel, waste oil and α-tocopherol in diets on apparent nutrient digestibility and AME in broiler chickens. SEM standard error of mean, P-value probability values. a :b Means in columns with the same superscript do not differ significantly P < 0.05. www.nature.com/scientificreports/ who indicated that the addition of 4% waste oil in diets reduced digestibility. Tavárez et al. 10 stated that the supplementation of waste oil in the diet diminished the growth performance of broilers. Moreover, feeding of waste oil results in lower digestibility of fat and energy in birds which is determined by the decreased capability of digestion 37 . It seems that parallel reduction of apparent CF digestibility, a reduced AME has been observed, indicating extensive fatty acid polymerization and a negative impact on the gastrointestinal tract 38 .

Main effect
Intestine microbial population. The effect of dietary pomegranate peel powder was significant (p < 0.05) on the lactobacillus and coliform bacteria populations from 11 to 49 days ( Table 8). The highest lactobacillus count was observed in 4% pomegranate peel powder groups (p < 0.05). Although, broilers fed on diets including 4% of pomegranate peel powder had significantly lower coliform populations than other groups (p < 0.05).
Although coliforms and the total bacteria population were not affected by dietary waste oil, significant differences were determined in the lactobacilli population of waste oil by linear contrast (p < 0.05) ( Table 7). Hence, supplementation of 4% waste oil in the diet significantly decreased the lactobacilli population in broilers (p < 0.05).
The lactobacilli bacteria populations were influenced by α-tocopherol supplementation within diets (p < 0.05) ( Table 9). The highest lactobacilli bacteria population was found in the group containing 200 mg/kg α-tocopherol (p < 0.05). Besides, the lowest lactobacilli bacteria population was observed in the non-supplemented group. Further, an interaction was not found between dietary treatments on the intestinal microbial population in this experiment. The present study concluded the optimal level of pomegranate peel in diets required to achieve the best intestinal health in broilers at the end of the experiment. The use of 4% pomegranate peel powder in diets increased lactobacilli bacteria count in broilers, whereas broilers fed with 4% pomegranate peel powder had lower coliform bacteria count. In similar studies, researchers have reported that pomegranate peel increased beneficial microorganisms of the intestine such as lactobacilli bacteria by restraining the pathogens bacteria, which improved digestion and absorption of broilers 39 . Overall, researchers suggested that phenolic compounds have antimicrobial characteristics. It was mentioned that phenolic compounds through the adhesion of pathogenic microorganisms by the 'lectin-receptor' mechanism and inhibition of the incorporation of pathogenic bacteria to the mucosal layer decreased gut pathogenic bacteria counts 40 . The phenolic compounds played an antimicrobial role against pathogenic microorganisms such as E.coli bacteria 41 . In general, it has been noted that higher lactobacillus counts in the intestine were well correlated with lower coliforms and salmonella bacteria count, which improved broiler performance and health 16 . The development in the lactobacilli bacteria count in this study seems to be related to the reduction in pathogenic bacteria in the gut 16 . Similarly, the tannins reduce pathogenic bacteria; hinder harmful gut microbe's metabolism and the activities of harmful microbial enzymes by preventing oxidative phosphorylation 42 . The results showed that the use of 4% waste oil in diets decreased the lactobacilli bacteria counts in broilers. Also, the inclusion of the different levels of waste oil in diets did not affect coliform bacteria and the total bacteria population. In the same case, it has been demonstrated that oxidative stress harmed intestinal microflora, which increased pathogenic bacteria in the intestine 43 . Oxidative stress influences the gut epithelial cells and stimulates intestinal bacteria and lipopolysaccharide (LPS). It seems that LPS is understood to cause apoptosis and damage in different cells 44 . Also, the researchers stated that oxidative stress could have a destructive effect on the intestinal immune barrier that increased pathogenic bacteria and inflammatory infiltrate 12 . In the present study, the supplementation of 200 mg/kg α-tocopherol was shown to increase the lactobacilli population in broilers. In contrast, Dalia et al. 45 reported that dietary 100 mg/kg α-tocopherol in broilers could not affect the caecal lactobacilli bacteria population. Results on the influence of α-tocopherol on microbial population are limited. Nevertheless, the researcher indicated that the decline in Salmonella spp. population due to α-tocopherol use unclear 45 . Hernken et al. 46 concluded that α-tocopherol could affect the antioxidant and immunity response of broilers. The use of vitamin E has been shown to decrease some of the adverse effects of oxidative stress 47 . Therefore, it is well known that the antioxidants substances improved intestinal health and functioning due to a reduction in oxidation stress in the intestine 45 .
Intestine morphology. These results (Table 9) indicate that the use of pomegranate peel powder affected villus height, crypt depth, and villus/crypt in broilers at 49 days (p < 0.05). In broilers, the villus height and villus/crypt significantly decreased in the group treated with 8% pomegranate peel powder compared with broilers fed diets with 4% pomegranate peel powder and non-supplemented group in all parts (duodenum, jejunum, and ileum) of the intestine. Also, the use of 8% pomegranate peel powder in diets significantly decreased crypt depth in broilers at 49 days in jejunum parts of the intestine (p < 0.05). A significant effect in villus height and villus/crypt at duodenum and jejunum was observed in the broilers that fed oxidized oil (p < 0.05). Additionally, the results of this study showed that the supplement of 4% oxidized oil in broilers significantly decreased villus height and villus/ crypt at the duodenum and jejunum of the intestine (p < 0.05). In general, no interaction among pomegranate peel powder, waste oil, and α-tocopherol on intestinal morphology has been observed in this study. The current research revealed that the 8% pomegranate peel powder use in diets reduced the villus height at all parts of the intestine, also the difference was significant just between 8% pomegranate peel powder and other groups. In the same manner, using 8% pomegranate peel powder in broiler diets decreased crypt depth in this study. Moreover, feeding 8% of pomegranate peel powder reduced the villi/crypts in the intestine. There is little information about pomegranate peel and intestinal morphology. It seems that these changes are defined by the high amount of tannins that inhibit nutrient absorption and reduce the growth of the body and other tissue in chickens 15,32,43 . It mentioned that the intestine is largely responsible for digestion and absorption of all the principal nutrients, consequently, lower villi height may be related to low digestive capacity 5 . In contrast with our results, researchers indicated that dietary pomegranate peel in quail diets had increased villus height in the jejunum of the intestine 5 . www.nature.com/scientificreports/ In this study, 4% waste oil inclusion in diets decreased the broilers' villus height and villi/crypts ratio at 30 and 60% parts of the intestine. This conclusion was comparable with the finding that high peroxide value oil decreased the villus high in birds 6 . The use of high peroxide value oil on intestine morphology was examined by other researchers 43 , who have shown that dietary oxidized oil decreases intestinal mucosa of birds in response to oxidative stress, which can result in low villus height. Similarly, Marchini et al. 48 observed that oxidative stress in the intestine reduced the villus height on broilers. It has been shown that birds under oxidative stress present smaller crypt depth, mucous area, and villus height of the small intestine, leading to an adverse influence on Table 8. The effects of using pomegranate peel powder, waste vegetable oil and α-tocopherol in diets on gut microbial population in broiler chickens. a,b ,c Means in columns with the same superscript do not differ significantly P < 0.05. SEM standard error of means, P-value probability values.

Main effect
Pomegranate peel (%) Meat quality. The effects of using different levels of pomegranate peel, waste oil, and α-tocopherol in the diet on the peroxide value of meat in broiler chickens are shown in Fig. 1. Results of the experiment indi- www.nature.com/scientificreports/ cated that the PV of meat was affected by dietary pomegranate peel and waste oil at the end of the experiment (p < 0.05). The inclusion of 4% pomegranate peel in diets decreased the meat PV of broilers compared to the control groups (p < 0.05). Dietary inclusion of 4% waste oil in diets increased the meat PV when compared to the control group (p < 0.05). No significant interactions have been observed for the meat quality profile. The inclusion of 4% pomegranate peel in diets decreased meat PV and agrees with Ghasemi-Sadabadi et al. 2 and Abdel Baset et al. 49 . It seems that the inhibitory influence of pomegranate peel on meat oxidation could be due to phenolic compounds, which have an antioxidant influence on lipid oxidation 49 . Similarly, Kishawy et al. 50 indicated that the use of pomegranate peel extract in diets increased the total phenol and total flavonoid contents of breast muscle in broilers. It can be concluded that the inclusion of 4% waste oil in diets improved the PV of meat. Researchers also confirmed that dietary waste oil enhanced the PV in the meat of broiler chickens 2,11,51 . Thus, the higher PV of meat has been indicated to be positively correlated with oxidized oil in diets. It had been stated that oxidative damage reduced the quality of poultry meat, including its PV 11 .

Conclusion
In conclusion, the dietary inclusion of 8% pomegranate peel reduced the growth performance of birds in comparison with the inclusion of 4% pomegranate peel and the control group. The inclusion of 4% pomegranate peel into broiler diets showed no significant effect in comparison with the control group.
The use of 8% pomegranate peel decreased apparent nutrient digestibility in this experiment. Also, the supplementation of 4% waste oil in the diet decreased CF and AME digestibility in broilers. The 4% pomegranate peel and 200 mg/kg α-tocopherol dietary inclusion improved the intestinal population in this experiment. The presence of waste oil in diets reduced the lactobacilli population. 4% pomegranate peel in the diet improved the gut morphology of broilers compared with the inclusion of 8% pomegranate peel and the control group. Whereas, the supplementation of 4% waste oil showed lower villus height and crypt depth compared to the other groups. In general, interaction effects among pomegranate peel powder, waste oil, and α-tocopherol have not been observed in this study. The effect of 4% pomegranate peel was decreased on the PV of meat. Also, the inclusion of 4% waste oil into diets reduced meat quality compared with other groups. alpha-tocopherol (200 mg/ kg) resulted in improved meat quality.

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.